This project represents a continued study of the biochemical cytology of several amitochondriate parasites of humans, primarily as it relates to their energy metabolism. These are Giardia lamblia, a cosmopolitan waterborne pathogen causing intestinal problems, Trichomonas vaginalis, a sexually transmitted parasite causing vaginitis and urethritis, Entamoeba histolytica, causative agent of amebic dysentery, and for comparative purposes, other trichomonads, and anaerobic ciliates and fungi of the rumen of ruminants. These organisms lack mitochondria, have a fundamentally O/2-independent energy metabolism and possess a pyruvate decarboxylating enzyme, pyruvate:ferredoxin oxidoreductase, that is not a homolog of the corresponding mitochondrial enzyme. These organisms represent two major types of metabolic organization. G. lamblia and E. histolytica belong to Type 1, which lacks organelles of energy metabolism, while T. vaginalis and the rumen protists are Type 2 organisms with hydrogenosomes, organelles of pyruvate oxidation leading to hydrogen formation. The project aims at furthering our understanding of the nature and evolutionary origin of the anaerobic mode of life in the organisms studied. Studies in the next project period will focus on biochemical characterization and comparative phylogenetic evaluation of enzymes of core metabolism. One group of enzymes studied will be those which differentiate amitochondriate protists from mitochondriate ones, e.g. pyruvate:ferredoxin oxidoreductase and those which differentiate Type 1 amitochondriate organisms from Type 2 ones, e.g., pyruvate dikinase and acetate thiokinase (ADP-forming). Another group includes enzymes which in view of available extensive comparative information can provide insight into the evolutionary process, e.g., adenylate kinase and glyceraldehyde- 3-phosphate dehydrogenase. Biochemical properties will be studied on enzymes purified from the organisms or, alternatively, obtained by hetero- logous expression. Primary structures will be determined by sequencing of genes cloned from gene libraries. A broader taxonomic coverage will be obtained by sequencing PCR products based on the use of degenerate primers and gDNA or cDNA of organisms not amenable to axenic cultivation. The sequence will be analyzed for correlations with the properties of the enzymes and for their evolutionary history.